Human gastric cancer tissue collection.
Gastric cancer tissues (n=6) in this study were obtained from patients with gastric cancer from First Affiliated Hospital, Anhui Medical University, Hefei, China. All patients signed informed consent to take part in the study. The study protocol was approved by the ethics boards of Anhui Medical University(Ethics:LLSC20150030), and tissue specimen acquisition was performed in accordance with the institutional guidelines. The written informed consent was obtained from all subjects.
Reagents.
Fetal bovine serum (FBS) was purchased from Millipore (Billerica, MA, USA). High-glucose DMEM medium was purchased from Hyclone (Logan, Utah, USA). Rabbit anti-C-Myc and rabbit anti-Cyclin D1 monoclonal antibodies were purchased from Cell Signaling (Danvers, MA, USA), Rabbit anti-MMP-9 monoclonal antibody was purchased from Millipore (Billerica, MA, USA), Rabbit anti-ZEB1 monoclonal antibody was purchased from Abcam (Eugene, USA), and anti-MMP-3 and anti-β1-actin monoclonal antibodies were purchased from Bioworld (Shanghai, China).
Cell culture
SGC-7901 cells and SGC-7901/DDP cells were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in cell culture flasks in DMEM (Hyclone, USA) supplemented with 10 % (v/v) heat-inactivated FBS (Millipore, USA), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (both from Beyotime, China). Cell cultures were maintained at 37 °C at an atmosphere of 5 % CO2.
Cells transient transfection of miR-200b and small interfering RNA silencing
SGC-7901/DDP cells were transfected with 100 nM of miR-200b inhibitor, miR-200b mimics (Biomics, USA), small interfering RNA (RNAi) of ZEB1 (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions.
Cell cycle analysis
To analyze the intracellular DNA content, SGC-7901/DDP cells were fixed in 70 % ethanol at 4 °C overnight. After then, cells were stained with 0.5 ml of propidium iodide (PI) staining buffer (Beyotime, China) containing 200 mg/ml RNase A, 50 μg/ml PI, at room temperature for 30 min in dark. Flow cytometric analysis was performed on Beckman Coulter.
Quantitative real-time PCR (q-PCR)
Total RNA was extracted from cultured SGC-7901/DDP cells by using RISO RNA Isolation Reagent (Biomics, USA) according to the manufacture's protocol. Expression of miR-200b was measured using an EzOmics miRNA qPCR Detection Primer Set (Biomics, USA) and EzOmics One-Step qPCR Kit (Biomics, USA) in PikoReal 96 real-time PCR system (Thermo, USA). The fold-change for miR-200b relative to U6 was calculated through the 2-△△CT method. Quantitative real-time PCR analyses of relative levels of C-Myc, CyclinD1, MMP-3, MMP-9, ZEB1 and β-actin mRNA were determined using the QuantiFast SYBR Green RT-PCR kits (QIAGEN, Germany) according to the manufacturer’s instruction. The sequences of primer pairs used were as follows: C-Myc (forward: 5’-GGACTATCCTGCTGCCAAGA-3’; reverse: 5’-CGCC TCTTGACATTCTCCTC-3’), CyclinD1 (forward: 5’-GATCAAGTGTGACCCGGA CT-3’; reverse: 5’-TCCTCCTCTTCCTCCTCCTC-3’), MMP-3 (forward: 5’-GGCCA GGGATTAATGGAGAT-3’; reverse: 5’-TGAAAGAGACCCAGGGAGTG-3’), MMP-9 (forward: 5’-GTACCACGGCCAACTACGAC-3’; reverse: 5’-GCCTTGGA AGATGAATGGAA-3’) and β-actin (forward: 5’-GCCAACACAGTGCTGTCTGG- 3’; reverse: 5’-AGGAGGAGCAATGATCTTG-3’).
Western blot
Cultured SGC-7901/DDP cells were lysed with lysis buffer (Beyotime, China) for Western blotting. Rabbit monoclonal antibodies directed against ZEB1 was used at 1:800, C-Myc, CyclinD1, MMP-3 and MMP-9 were used at 1:500, and β-actin was used at 1:1000. After incubated with primary antibodies, the nitrocellulose blots were washed for four times in TBS/Tween-20 before incubation in goat anti-mouse or anti-rabbit HRP-conjugating antibody at 1:10000 diluted in TBS/Tween-20 containing 5 % skim milk for 1 h. After washing four times with TBS/Tween-20 the protein blots were detected using the ECL-chemiluminescent kit (ECL-plus, Thermo Scientific).
Luciferase reporter assays
SGC-7901/DDP cells were cultured into 24-well plates, and transfected transiently with 200 ng DNA of ZEB1 3’UTR WT plasmid, 60 nmol of miR-200b mimics or the negative controls (Gene Pharma, Shanghai), using 2.25 μl of lipofectamine 2000 and 100 μl of Opti-MEM (Invitrogen, USA). The cells were harvested and lysed 48 h post- transfection. The luciferase activities were measured consecutively by the Dual- Luciferase Reporter 1000 Assay system (Promega, USA).
Wound-healing analysis
SGC-7901/DDP cells were cultured 2 days in 24-well plate (5 × 105/ml cells/well) in DMEM with 20 % FBS. And then, the cells were serum deprived and scratched. 24 h later Type II alveolar epithelial cells were fixed with methanol, stained with crystal violet stain, and viewed under an Olympus BX-51 microscope.
Statistical analysis.
Data were presented as means ± SD and analyzed using SPSS16.0 software. Statistical significances were determined by one-way ANOVA with the post-hoc Dunnett's test. In all assays, values of P < 0.05 were considered to be statistically significant.